CN110954518A

CN110954518A - Preparation method of dumbbell-type DNA/copper nanoparticle fluorescence biosensor and application of dumbbell-type DNA/copper nanoparticle fluorescence biosensor in quantitative determination of ATP

Info

Publication number: CN110954518A
Application number: CN201911273379.2A
Authority: CN
Inventors: 王广凤; 陈纪华
Original assignee: Anhui Normal University
Current assignee: Anhui Normal University
Priority date: 2019-12-12
Filing date: 2019-12-12
Publication date: 2020-04-03
Anticipated expiration: 2039-12-12
Also published as: CN110954518B

Abstract

The invention discloses a preparation method of a dumbbell-shaped DNA/copper nanoparticle fluorescence biosensor and application thereof in quantitative detection of ATP, wherein the dumbbell-shaped DNA is prepared by carrying out preparation on DS DNA phosphorylated at the 5' end under the action of T4DNA ligase and ATP, the dumbbell-shaped DNA/copper nanoparticle fluorescence biosensor combined with copper-sodium rice grains is further prepared on the basis of the dumbbell-shaped DNA, and the fluorescence intensity of the dumbbell-shaped DNA/copper nanoparticle fluorescence biosensor is increased along with the increase of the concentration of added adenosine triphosphate, so that a linear relation between the concentration of ATP and the fluorescence intensity is constructed, the high-sensitivity and specific detection of the adenosine triphosphate is realized, and the dumbbell-shaped DNA/copper nanoparticle fluorescence biosensor has the advantages of simple operation, high sensitivity and low detection limit.

Description

Preparation method of dumbbell-type DNA/copper nanoparticle fluorescence biosensor and application of dumbbell-type DNA/copper nanoparticle fluorescence biosensor in quantitative determination of ATP

Technical Field

The invention belongs to the technical field of fluorescent sensing preparation, and particularly relates to a preparation method of a dumbbell-shaped DNA/copper nanoparticle fluorescent biosensor and application of the dumbbell-shaped DNA/copper nanoparticle fluorescent biosensor in quantitative detection of ATP.

Background

Adenosine Triphosphate (ATP) is a multifunctional nucleotide that is not only a universal energy source, but is also an extracellular signaling mediator, involved in many biological processes including membrane ion channels, DNA replication, biosynthesis, and is also used as an indicator of cellular activity and cellular damage in organisms. Therefore, it has wide applications in biochemical research and clinical diagnosis, and is essential for highly sensitive and selective detection of ATP. Strategies for ATP detection have been developed, and many methods based on host-guest, peptide, conjugated polymer, DNA/RNA aptamers and ATP-dependent ligation reactions, even though they show very good analytical performance, usually involve signal labeling, and are relatively long and costly. Based on the above, we have experimentally explored and proposed an optimal light-emitting dumbbell-type (DS) DNA template structure for growing CuNPs, which is used as a fluorescent probe to test the high-sensitivity detection of ATP.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method of a dumbbell-shaped DNA/copper nanoparticle fluorescence biosensor and application thereof in quantitative detection of ATP, wherein the dumbbell-shaped DNA is prepared by the DS DNA phosphorylated at the 5' end under the action of T4DNA ligase and ATP, and the dumbbell-shaped DNA/copper nanoparticle fluorescence biosensor combined with copper sodium particles is further prepared on the basis of the dumbbell-shaped DNA, so that high-sensitivity detection of ATP can be realized.

The technical scheme adopted by the invention is as follows:

a preparation method of a dumbbell-shaped DNA/copper nanoparticle fluorescence biosensor comprises the following steps:

(1) putting the DS DNA solution with 5' end phosphorylation in a buffer solution, then adding T4DNA ligase and ATP, and reacting for 50-60 min to obtain a dumbbell type DNA solution;

(2) adding the dumbbell-shaped DNA solution obtained in the step (1) into a buffer solution, and then adding Cu²⁺And uniformly mixing the solution and the sodium ascorbate solution, and reacting for at least 5min to obtain the dumbbell-shaped DNA/copper nanoparticle fluorescence biosensor.

The gene sequence of the 5' phosphorylated DS DNA is as follows:

5’-PO₄-ATATATATATATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATATATATATATATATATATATATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATATATATATAT-3’。

the step (1) is followed by: exo I and Exo III were added to the dumbbell DNA solution, reacted for 60min, and then incubated at 80 ℃ for 5 min. The dumbbell-shaped DNA prepared by the invention is proved not to be damaged by Exo I and Exo III through the addition of the Exo I and Exo III.

In the step (1), the volume ratio of the DS DNA solution with 5' end phosphorylation, the buffer solution, the T4DNA ligase and the ATP is 50:144:1: 5; the concentration of the 5' end phosphorylated DS DNA solution is 1 μ M; the concentration of the T4DNA ligase was 350U/. mu.L.

The buffer solution was a 10mM MOPS buffer solution at PH 7.6.

In the step (2), the dumbbell type DNA solution, the buffer solution and the Cu are used²⁺The volume ratio of the solution to the sodium ascorbate solution is 1:1:1: 1; the concentration of the dumbbell type DNA solution is 1 mu M; the Cu²⁺The concentration of the solution was 0.8 mM; the concentration of the sodium ascorbate solution is 8 mM.

Further, the volume ratio of the 5' -phosphorylated DS DNA, Exo I and Exo III was 10:1: 1.

The invention also provides application of the dumbbell type DNA/copper nanoparticle fluorescence biosensor prepared by the preparation method in quantitative detection of ATP.

The ATP quantitative detection method comprises the following steps:

(a) respectively putting the DS DNA solution with 5' end phosphorylation in a buffer solution, then respectively adding T4DNA ligase and ATP with different concentrations, and reacting for 50-60 min to obtain a dumbbell type DNA solution;

(b) respectively adding the dumbbell-shaped DNA solution obtained in the step (1) into a buffer solution, and then adding Cu²⁺Uniformly mixing the solution and the sodium ascorbate solution, and reacting for at least 5min to respectively obtain the copper nanoparticle fluorescence biosensor;

(c) respectively testing the fluorescence intensity of each copper nanoparticle fluorescence biosensor obtained in the step (b), and drawing a standard curve by taking the ATP concentration as a horizontal coordinate and the fluorescence intensity value of each copper nanoparticle fluorescence biosensor at 630nm as a vertical coordinate to obtain a linear equation;

(d) and (3) according to a standard curve or a linear equation, calculating the concentration of the ATP to be detected according to the fluorescence intensity of the copper nanoparticle fluorescence biosensor prepared by repeating the steps (a) and (b) on the basis of the ATP to be detected.

In the step (c), the excitation wavelength and the emission wavelength of the fluorescence spectrophotometer are set to be 340nm and 630nm respectively.

Based on the connection effect of T4DNA ligase and ATP, the 5 'end phosphorylated sticky end can be connected with the 3' end to form dumbbell-shaped DNA under the condition that target adenosine triphosphate exists, and when exonuclease I and exonuclease III coexist, the dumbbell-shaped DNA template is kept intact and cannot be damaged. And under the existence of sodium ascorbate and copper ions, copper nanoparticles can be generated on the dumbbell-shaped DNA template to construct a dumbbell-shaped DNA/copper nanoparticle fluorescence biosensor, and the fluorescence intensity of the dumbbell-shaped DNA/copper nanoparticle fluorescence biosensor is increased along with the increase of the concentration of added adenosine triphosphate, so that a linear relation between the ATP concentration and the fluorescence intensity is constructed, the high-sensitivity and high-specificity detection of the adenosine triphosphate is realized, and the method has the advantages of simple operation, high sensitivity and low detection limit.

Compared with the prior art, the preparation method of the dumbbell-shaped DNA/copper nanoparticle fluorescence sensor provided by the invention has the advantages that the used DS DNA with 5' end phosphorylation forms dumbbell-shaped DNA under the action of DNA ligase and ATP, the operation is simple, the cost is very low, any chemical labeling and modification are avoided, and the background signal is low.

Drawings

FIG. 1 is a schematic diagram of a dumbbell-shaped DNA/copper nanoparticle fluorescence biosensor;

FIG. 2 is a fluorescence spectrum of dumbbell-shaped DNA/copper nanoparticles constructed under different ATP concentrations, with the final ATP concentrations corresponding to a-k being 0.1, 1, 10, 20, 50, 100, 200, 500, 1000, 5000, and 10000nM, respectively;

FIG. 3 is a linear relationship between ATP concentration and fluorescence intensity of dumbbell-shaped DNA/copper nanoparticles;

FIG. 4 is a diagram showing the feasibility of dumbbell-shaped DNA/copper nanoparticles for ATP detection, wherein a represents DS DNA +100nM ATP + T4 ligase + Exo I + Exo III; b represents DS DNA + Exo I + Exo III; c represents DS DNA + T4 ligase + Exo I + Exo III;

FIG. 5 is a fluorescence spectrum of fluorescent CuNPs prepared from hairpin DNA numbered 1-4;

FIG. 6 is a fluorescence spectrum of fluorescent CuNPs prepared from hairpin DNA numbered 5-8;

FIG. 7 is a schematic structural diagram of fluorescent CuNPs templates prepared from hairpin DNAs numbered 1-4;

FIG. 8 is a schematic structural diagram of fluorescent CuNPs templates prepared from hairpin DNAs numbered 5-8;

FIG. 9 is a fluorescence spectrum of the formation time of dumbbell-shaped DNA/copper nanoparticles;

FIG. 10 is a fluorescence spectrum of the effect of sodium ascorbate concentration on the fluorescence of dumbbell-shaped DNA/copper nanoparticles;

FIG. 11 is a fluorescence spectrum of the effect of copper ion concentration on the fluorescence of dumbbell-shaped DNA/copper nanoparticles;

FIG. 12 is a fluorescence spectrum of the effect of different pH values on the fluorescence of dumbbell-shaped DNA/copper nanoparticles;

FIG. 13 is a fluorescence spectrum of different analytes on dumbbell-shaped DNA/copper nanoparticles: blank, GTP, CTP, UTP, ATP and mixtures thereof;

FIG. 14 is a graph showing the effect of T4DNA ligase concentration on the fluorescence spectra of dumbbell-shaped DNA/copper nanoparticles;

FIG. 15 is a fluorescence spectrum of the effect of T4DNA ligase ligation time on dumbbell-shaped DNA/copper nanoparticles.

Detailed Description

The invention is described in detail below with reference to the following examples and the accompanying drawings.

Example 1

(1) dissolving 50 μ L of 1 μ M DS DNA solution phosphorylated at the 5' end in 34 μ L of 10mM MOPS buffer solution with PH 7.6, then adding 1 μ L of 350U/μ L T4DNA ligase and 5 μ L ATP, and supplementing 10mM MOPS buffer solution with PH 7.6 until the volume of the reaction system is 200 μ L, and at room temperature, 50 minutes can generate ATP-initiated ligation reaction to obtain dumbbell DNA solution;

the gene sequence of the 5' end phosphorylated DS DNA is as follows:

5’-PO₄-ATATATATATATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATATATATATATATATATATATATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATATATATATAT-3’；

(2) mu.L of the dumbbell DNA solution obtained in step (1) was added to 50. mu.L of a 10mM MOPS buffer solution having a pH of 7.6, and 50. mu.L of 0.8mM CuSO was added₄And (3) uniformly mixing the solution and 50 mu L of 8mM sodium ascorbate solution, reacting for 6min to obtain the dumbbell-type DNA/copper nanoparticle fluorescence biosensor, wherein the fluorescence intensity of the dumbbell-type DNA/copper nanoparticle fluorescence biosensor is sequentially increased along with the increase of the ATP concentration in the step (1), and the result is shown in figure 2, so that the quantitative detection of ATP can be realized.

Example 2

The other steps are the same as example 1, except that ATP with different concentrations is added in the step (1) respectively, and the final concentrations of ATP in the reaction system are respectively 0.1, 1, 10, 20, 50, 100, 200, 500, 1000, 5000 and 10000 nM;

and (3) respectively testing the fluorescence intensity of each dumbbell-type DNA/copper nanoparticle fluorescence biosensor obtained in the step (2), wherein the excitation wavelength and the emission wavelength of a fluorescence spectrophotometer are respectively 340nm and 630 nm. As shown in fig. 2, the ATP concentration in the range of 0 to 20nM is used as the abscissa, the fluorescence intensity value of each dumbbell-shaped DNA/copper nanoparticle fluorescence biosensor at 630nM is used as the ordinate to construct a standard curve, and as shown in fig. 3, the linear equation F is 27.06C_ATP+874.09 with a correlation coefficient R²＝0.992；

And (3) according to a standard curve or a linear equation, calculating the concentration of the ATP to be detected according to the fluorescence intensity of the dumbbell-type DNA/copper nanoparticle fluorescence biosensor prepared in the step (1) and the step (2) repeated for the ATP to be detected.

Example 3

To verify the stability of the dumbbell-type DNA solution, the same procedure as in example 1 was repeated except that the final ATP concentration in the fixing step (1) was 100nM, 5. mu.L of 5U/. mu.L Exo I and 5. mu.L of 200U/. mu.L Exo III were added to the dumbbell-type DNA solution obtained in step (1) of example 1, and the mixture was sheared at room temperature for 60 minutes, and then incubated at 80 ℃ for 5 minutes after 60 minutes to terminate the degradation reaction. Then 50. mu.L of 8mM sodium ascorbate was added to the above mixture, mixed well and 50. mu.L of 0.8mM CuSO₄And adding the solution, fully mixing, detecting the fluorescence intensity of a reaction system at room temperature for 6 minutes, and finding that the obtained product, namely the prepared biosensor still has strong fluorescence, which indicates that the dumbbell-shaped DNA structure cannot be sheared by exonuclease, and when copper ions and ascorbic acid exist, fluorescent copper nanoparticles still can be formed and have strong fluorescence intensity, as shown by a curve a in figure 4.

Under the same conditions, 50. mu.L of 1. mu.M 5' -phosphorylated DS DNA solution was dissolved in 150. mu.L of 10mM MOPS buffer solution having pH 7.6, 5. mu.L of 5U/. mu.L Exo I and 5. mu.L of 200U/. mu.L Exo III were added, the mixture was sheared at room temperature for 60 minutes, the mixture was incubated at 80 ℃ for 5 minutes after 60 minutes, 50. mu.L of 8mM sodium ascorbate was added to the mixture, the mixture was thoroughly mixed, and 50. mu.L of 0.8mM CuSO was added₄Adding the solution, fully mixing, and testing the fluorescence intensity of the system at room temperature for 6 minutes, wherein the result is shown as a curve b in figure 4;

under the same conditions, 50. mu.L of 1. mu.M of 5' -phosphorylated DS DNA solution was dissolved in 149. mu.L of 10mM MOPS buffer solution having pH 7.6, 1. mu.L of 350U/. mu. L T4DNA ligase was added thereto, the reaction was carried out for 50 minutes, 5. mu.L of 5U/. mu.L Exo I and 5. mu.L of 200U/. mu.L Exo III were further added thereto, the mixture was sheared at room temperature for 60 minutes, the mixture was incubated at 80 ℃ for 5 minutes after 60 minutes, 50. mu.L of 8mM sodium ascorbate was added to the mixture, the mixture was thoroughly mixed, and 50. mu.L of 0.8mM CuSO was further added thereto₄Adding the solution, fully mixing, and testing the fluorescence intensity of the system at room temperature for 6 minutes, wherein the result is shown as a curve c in figure 4;

as can be seen from the curves b and c in FIG. 4, when no ATP is added, the 5 'end and the 3' end of the DS DNA with 5 'end phosphorylation are not connected, and the DS DNA template is sheared under the action of the exonuclease, and no template is used for growing the copper nanoparticles, so that the fluorescent copper nanoparticles are not formed, which shows that the 5' end and the 3 'end of the DS DNA with 5' end phosphorylation are connected only in the presence of ATP, so that the DS DNA template is not damaged by the exonuclease, and the copper nanoparticle template is provided.

Example 4

Screening of the respective reaction conditions

4.1DNA template optimization

(1) Mu.l of each of eight 1. mu.M hairpin DNA (Nos. 1, 2, 3, 4, 5, 6, 7, and 8) solutions was added to 98. mu.l of a MOPS buffer solution (pH: 7.610 mM), and incubated at 95 ℃ for 5 minutes to uncoil the hairpin DNA and culture the same to obtain a mixed solution; the preparation method of the hairpin DNA solution comprises the following steps: the hairpin DNA sequence was dissolved in 10mM MOPS PH 7.6 buffer solution to a concentration of 1 μ M;

(2) to the eight hairpin DNA solutions prepared in step (1), 50. mu.L of 0.8mM CuSO was added₄The solution and 50. mu.L of 8mM sodium ascorbate solution were incubated for 10 minutes, and the fluorescence intensity of each reaction system was measured, and as shown in FIGS. 5 and 6, it was found that the hairpin DNA of No. 5 had the highest fluorescence intensity and thus the optimal luminophore was obtained, and therefore the gene sequence of the selected DS DNA phosphorylated at the 5' end was:

5’-PO₄ATATATATATATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATATATATATATATATATATATATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATATATATATAT-3' to construct dumbbell DNA by ligation of T4DNA ligase and ATP.

Schematic structural diagrams of the fluorescence-passing nanoparticle templates formed in each reaction system are shown in fig. 7 and 8. The gene sequence of each hairpin DNA is shown below:

DNA1, No. 1:

3’-GCGCGCGCGCGCGCGCGCGCGCGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCGCGCGCGCGCGCGCGCGCGCGC-5’；

DNA2, No. 2:

3’-GCGCGCGCGCGCGCGCGCGCGCGCTTTTTTTTTTTTTTTCCCCCTTTTTTTTTTTTTTTGCGCGCGCGCGCGCGCGCGCGCGC-5’；

DNA3, No. 3:

3’-GCGCGCGCGCGCGCGCGCGCGCGCTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCTTTTTTTTTTTTTTTGCGCGCGCGCGCGCGCGCGCGCGC-5’；

DNA4, No. 4:

3’-GCGCGCGCGCGCGCGCGCGCGCGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCGCGCGCGCGCGCGCGCGCGCGCGC-5’；

DNA5, No. 5:

3’-ATATATATATATATATATATATATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATATATATATATATATATATATAT-5’；

DNA6, No. 6:

3’-ATATATATATATATATATATATATGCGCGCGCGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCGCGCGCGCATATATATATATATATATATATAT-5’；

DNA7, No. 7:

3’-ATATATATATATATATATATATATGCGCGCGCGCGCGCGCGCGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCGCGCGCGCGCGCGCGCGCATATATATATATATATATATATAT-5’；

DNA8, No. 8:

3’-ATATATATATATATATATATATATGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCATATATATATATATATATATATAT-5’。

4.2 optimization of the reaction conditions

The formation time fluorescence spectrum of the dumbbell-shaped DNA/copper nanoparticles is shown in FIG. 9, from which it can be seen that the fluorescence intensity of the reaction system has already stabilized up to 6min, indicating that a stable fluorescence sensor has been formed.

FIG. 10 shows the fluorescence spectrum of the effect of the final concentration of sodium ascorbate on the fluorescence of dumbbell-shaped DNA/copper nanoparticles, from which it can be seen that the fluorescence intensity of the reaction system is maximal at a final concentration of sodium ascorbate of 2mM, and thus the final concentration of sodium ascorbate is 2mM during the reaction.

FIG. 11 shows the fluorescence spectrum of dumbbell-shaped DNA/copper nanoparticles affected by the final concentration of copper ions, which shows that the final concentration of copper ions in the reaction system is 100mM because the fluorescence intensity of the reaction system is the greatest at 100mM, and thus the final concentration of copper ions is 100mM during the reaction.

As shown in FIG. 12, the fluorescence spectra of dumbbell-shaped DNA/copper nanoparticles affected by different pH values show that the fluorescence intensity of the reaction system is the greatest when the pH of the reaction system is 7.6, and thus the MOPS buffer solution used in the reaction has a pH of 7.6.

The fluorescence spectra of different analytes on dumbbell-shaped DNA/copper nanoparticles are shown in FIG. 13, and it can be seen from the figure that the reaction system can exclude the interference of other substances.

As shown in FIG. 14, it can be seen from the fluorescence spectrum of the effect of the final concentration of T4DNA ligase on dumbbell-shaped DNA/copper nanoparticles that the fluorescence intensity of the reaction system tends to be stable when the final concentration of T4DNA ligase is 1.75U/uL, so that the final concentration of T4DNA ligase added during the reaction is 1.75U/uL

As shown in FIG. 15, it can be seen from the fluorescence spectrum of the effect of the ligation time of T4DNA ligase on dumbbell-shaped DNA/copper nanoparticles that the fluorescence intensity of the reaction system tends to be stable 50min after T4DNA ligase is added, so that stable dumbbell-shaped DNA can be obtained 50min after T4DNA ligase and ATP are added.

Each of the above reaction conditions was an experiment conducted under the conditions of example 1 or 3, with the ATP concentration being fixed at 100nM, and with the conditions being varied individually.

The above detailed description of the preparation method of the dumbbell-shaped DNA/copper nanoparticle fluorescence biosensor and its application in quantitative detection of ATP with reference to the embodiments is illustrative and not restrictive, and several embodiments can be enumerated according to the limited scope, so that variations and modifications without departing from the general concept of the present invention shall fall within the protection scope of the present invention.

SEQUENCE LISTING

<110> university of teacher's university in Anhui

<120> preparation method of dumbbell type DNA/copper nanoparticle fluorescence biosensor and application of dumbbell type DNA/copper nanoparticle fluorescence biosensor in quantitative determination of ATP

In (1)

<130>1

<160>9

<170>PatentIn version 3.3

<210>1

<211>108

<212>DNA

<213>DS DNA

<400>1

5'-PO4-atatatatat attttttttt tttttttttt tttttttttt ttatatatat atatatatat 60

atatattttt tttttttttt tttttttttt ttttttatat atatatat-3' 108

<210>2

<211>78

<212>DNA

<213>DNA1

<400>2

5'-gcgcgcgcgc gcgcgcgcgc gcgctttttt tttttttttt tttttttttt ttttgcgcgc 60

gcgcgcgcgc gcgcgcgc-3' 78

<210>3

<211>83

<212>DNA

<213>DNA2

<400>3

5'-gcgcgcgcgc gcgcgcgcgc gcgctttttt tttttttttc cccctttttt tttttttttg 60

cgcgcgcgcg cgcgcgcgcg cgc-3' 83

<210>4

<211>93

<212>DNA

<213>DNA3

<400>4

5'-gcgcgcgcgc gcgcgcgcgc gcgctttttt tttttttttc cccccccccc cccctttttt 60

tttttttttg cgcgcgcgcg cgcgcgcgcg cgc-3' 93

<210>5

<211>108

<212>DNA

<213>DNA4

<400>5

5'-gcgcgcgcgc gcgcgcgcgc gcgctttttt tttttttttt tttttttttt ttttcccccc 60

cccccccccc cccccccccc ccccgcgcgc gcgcgcgcgc gcgcgcgc-3' 108

<210>6

<211>78

<212>DNA

<213>DNA5

<400>6

5'-atatatatat atatatatat atattttttt tttttttttt tttttttttt ttttatatat 60

atatatatat atatatat-3' 78

<210>7

<211>98

<212>DNA

<213>DNA6

<400>7

5'-atatatatat atatatatat atatgcgcgc gcgctttttt tttttttttt tttttttttt 60

ttttgcgcgc gcgcatatat atatatatat atatatat-3' 98

<210>8

<211>118

<212>DNA

<213>DNA7

<400>8

5'-atatatatat atatatatat atatgcgcgc gcgcgcgcgc gcgctttttt tttttttttt 60

tttttttttt ttttgcgcgc gcgcgcgcgc gcgcatatat atatatatat atatatat-3' 118

<210>9

<211>138

<212>DNA

<213>DNA8

<400>9

5'-atatatatat atatatatat atatgcgcgc gcgcgcgcgc gcgcgcgcgc gcgctttttt 60

tttttttttt tttttttttt ttttgcgcgc gcgcgcgcgc gcgcgcgcgcgcgcatatat 120

atatatatat atatatat-3' 138

Claims

1. A preparation method of a dumbbell-shaped DNA/copper nanoparticle fluorescence biosensor is characterized by comprising the following steps:

(1) putting the 5' end phosphorylated DSDNA solution in a buffer solution, then adding T4DNA ligase and ATP, and reacting for 50-60 min to obtain a dumbbell type DNA solution;

2. The method according to claim 1 or 2, wherein the gene sequence of the 5' -phosphorylated DSDNA is:

3. the method of claim 1, wherein step (1) is further followed by: exo I and Exo III were added to the dumbbell DNA solution, reacted for 60min, and then incubated at 80 ℃ for 5 min.

4. The method according to any one of claims 1 to 3, wherein in the step (1), the volume ratio of the 5' -end phosphorylated DSDNA solution, the buffer solution, the T4DNA ligase and the ATP is 50:144:1: 5; the concentration of the DSDNA solution with 5' end phosphorylation is 1 mu M; the concentration of the T4DNA ligase was 350U/. mu.L.

5. The method according to any one of claims 1 to 3, wherein the buffer solution is a 10mM MOPS buffer solution having a pH of 7.6.

6. A method according to any one of claims 1 to 3The preparation method of (1), wherein in the step (2), the dumbbell-shaped DNA solution, the buffer solution, and the Cu are mixed²⁺The volume ratio of the solution to the sodium ascorbate solution is 1:1:1: 1.

7. The method according to claim 3, wherein the volume ratio of the 5' -phosphorylated DS DNA, Exo I and Exo III is 10:1: 1.

8. The dumbbell-shaped DNA/copper nanoparticle fluorescence biosensor prepared by the preparation method according to any one of claims 1 to 7 is applied to quantitative determination of ATP.

9. The use according to claim 8, wherein the quantitative ATP detection method comprises the following steps:

(a) respectively putting DSDNA solution with 5' end phosphorylation in buffer solution, then respectively adding T4DNA ligase and ATP with different concentrations, and reacting for 50-60 min to obtain dumbbell type DNA solution;

10. Use according to claim 8, wherein in step (c) the excitation and emission wavelengths of the spectrofluorometer are set to 340nm and 630nm, respectively.